Aptamers are versatile, single stranded oligonucleotides that can be employed for the molecular recognition of different target structures via a strategy called Systematic Evolution of Ligands via EXponential enrichment (SELEX). Traditionally, aptamers were evolved against small molecules or proteins, and subsequently modified to enable application in a variety of in vitro assays. However, in recent years both the selection and application of aptamers has progressed towards considerably more complex targets such as cells, tissue slices or even live organisms. The target structures can be bound with high selectivity and affinity without knowledge of the actual molecule that is recognized by the aptamer. On the other hand, once an aptamer for targets of such complexity has been identified, this lack of knowledge immediately turns into a severe disadvantage, as it prevents further advancements, for example, as an analytical tool or as a biomarker for certain disease states. These restrictions require technologies that allow for the rational identification of unknown target molecules of an aptamer. Especially, the proteome-wide profiling or target identification of aptamers is not well established.
To identify aptamer targets photo-crosslinking of aptamers by photoreactive 5-iododeoxyuridines incorporated in an aptamer was used. However, the method suffers from a loss in affinity observed when introducing photoreactive nucleotides into an aptamer and requires tedious optimization of the position of the crosslinker moiety. Moreover, as this approach requires similar optimizations for other aptamer/target pairs, its general applicability is limited.
Therefore, the object underlying the present invention was to provide a method that allows for the identification of aptamer target structures. Especially it was an object of the present invention to provide a method that avoids optimization for each aptamer/target pair.